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WO2009129693A1 - Sondes, produits à base de puce liquide et procédés pour la détection de mutations du gène egfr - Google Patents

Sondes, produits à base de puce liquide et procédés pour la détection de mutations du gène egfr Download PDF

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Publication number
WO2009129693A1
WO2009129693A1 PCT/CN2009/000429 CN2009000429W WO2009129693A1 WO 2009129693 A1 WO2009129693 A1 WO 2009129693A1 CN 2009000429 W CN2009000429 W CN 2009000429W WO 2009129693 A1 WO2009129693 A1 WO 2009129693A1
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Prior art keywords
seq
exon
probe
microspheres
amino
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PCT/CN2009/000429
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English (en)
Chinese (zh)
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许嘉森
杨惠夷
林一群
徐军
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广州益善生物技术有限公司
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Publication of WO2009129693A1 publication Critical patent/WO2009129693A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention belongs to the field of molecular biology, and relates to medicine and biotechnology, and specifically relates to a detection probe, a liquid phase chip and a detection method thereof for a mutation site of an EGFR gene.
  • Epidermal growth factor receptor is a multifunctional glycoprotein widely distributed on the cell membrane of human tissues. It has tyrosine kinase activity and is one of the four members of the ErbB family. HER1 or ErbBl. Upon activation of the ligand, EGFR initiates intracellular signaling, undergoes a cascade of adaptor proteins and enzymes in the cytoplasm, regulates transcription of transcription factor-activated genes, and directs cell migration, adhesion, proliferation, differentiation, and apoptosis. Studies have shown that the high expression or abnormal expression of EGFR in many solid tumors provides a theoretical basis and experimental basis for EGFR-targeted tumor therapy and signal transduction intervention for EGFR signaling pathway.
  • EGFR receptor tyrosine kinase inhibitors gefitinib (Genfitinib, Irressa) and erlotinib (Erlotinib, Tarceva, Tarceva) have been approved by the US Food and Drug Administration for the treatment of advanced non-small cells. Lung cancer (NSCLC). In February 2005, China also approved gefitinib to enter the clinic. These drugs are used in the treatment of other tumors (such as breast cancer, colon cancer, and gastric cancer) and are now in clinical trials.
  • NSCLC Lung cancer
  • Gefitinib, erlotinib is an oral small molecule EGFR antagonist that competes with ATP for binding to the ATP-binding pocket of the EGFR tyrosine kinase domain to inhibit EGFR activity. It has been used in advanced and unsuitable for traditional chemotherapy. The clinical treatment of patients with non-small cell lung cancer is currently the most effective treatment for some patients with NSCLC. However, the patient's responsiveness to this molecularly targeted drug is related to the individual's genetic background. In June 2004, researchers at Harvard Medical School in the United States, Lynch et al. and Paez, etc., first reported that mutations in the EGFR tyrosine kinase coding region in lung cancer cells are a necessary prerequisite for targeted therapeutics.
  • the detection of the presence of relevant EGFR functional mutations can accurately predict the effectiveness of molecularly targeted drug therapy, facilitate clinical accurate drug use, and significantly improve drug efficacy. To maximize the benefit of patients, while avoiding the increase in medical expenses for patients and the waste of social medical resources caused by irrational use of drugs, reducing the aging loss of unnecessary treatment and economic losses.
  • the detection standard for EGFR (tyrosine kinase domain) gene mutations is the direct sequencing of tissue DNA samples.
  • the method was first reported by Lynch et al. and Paez et al.
  • the advantage is that the range and type of EGFR gene mutation can be accurately understood, and the relationship between gene mutation and the clinical efficacy of gefitinib has been affirmed.
  • gene sequencing as a clinical routine diagnosis method also has many obstacles to be excluded.
  • the detection of trace tissue specimens is difficult. Tissue DNA is amplified by PCR and then gene ten Sequencing, DNA requirements are at least 100 ng or more.
  • the specimens for detecting EGFR gene mutations are mainly taken from postoperative tissue samples.
  • a large amount of free nucleic acid (DNA or RNA) is present in the body fluid (plasma, serum or pleural effusion) of a tumor patient, and its content is about 10 times higher than that of a healthy person.
  • This type of free nucleic acid is mainly derived from apoptosis and necrosis of cancer cells, and its genetic characteristics are identical to those of tumor genomic DNA. Therefore, serum free nucleic acid can be used for detection of oncogene mutation.
  • free nucleic acids may have only a very small number of mutated genes, but a large number of wild-type genes, a large number of mixed wild-type genes will hinder the detection of mutant genes, thus requiring a highly sensitive and specific mutant detection method. To assess EGFR gene mutations.
  • the frequency of EGFR-related gene mutations is about 30%-50% in Asia, mainly in the base deletion mutation of 19 exons ( ⁇ 746-750 ⁇ , including two common types, See the table below) and the point mutation of the 21 exon (L858R).
  • the base deletion mutation of 19 exons accounted for about 54.5 % of the total number of mutations
  • the point mutation of 21 exons accounted for about 40.3 %
  • the present invention selects the 19 exons and 21 exons with the highest mutation rate for detection.
  • Exonl9 Common Missing Type Missing Length Exonl9 Sequence one indicates missing part
  • E746-A750 15bp GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAA (abbreviated as E19M1) AACATCTCCGAAAGCCAACAAGGAAATCCTCGAT del E746-A750 (2) 15bp GGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAA (abbreviated as E19M2) G ACATCTCCGAAAGCCAACAAGGAAATCCTCGAT
  • One of the objects of the present invention is to provide a probe sequence for the detection of mutations in the EGFR gene.
  • Probe for detection of mutations in the EGFR gene including
  • a wild-type probe selected from exonl9 exons selected from any one of SEQ ID N0.1 to SEQ IDN0.3, and an exo-expressed exonl9 selected from any one of SEQ ID N0.4 - SEQ ID N0.6
  • a del E746-A750 (1) mutant probe, Wo IV or a mutant del E746-A750 (2) probe selected from the exonl9 exon selected from any one of SEQ IDN 0.29 to SEQ IDN 0.31; and / or
  • a wild type probe selected from the group consisting of 3 £(510 ⁇ ).7 ⁇ 86 (3101»10.9 for the £0 ⁇ 1 exon, and selected from any one of SEQ ID NO. 10 to SEQ ID N0.12 A mutant probe directed against the Exon21 exon.
  • Another object of the present invention is to provide a liquid phase chip for detecting mutations in the EGFR gene.
  • the liquid phase chip can be used to detect related mutations in the exons 19 and 21 of the EGFR gene, and can be used to predict the therapeutic effects of gefitinib, erlotinib and the like.
  • a liquid phase chip for detecting EGFR gene mutation mainly comprising: a coated base sequence selected from the group consisting of SEQ ID NO: 1
  • the microspheres have different color coding
  • a primer for amplifying a target sequence having a 19 exon and/or 21 exon mutation site, and the end of the target sequence has a biotin label.
  • the spacer arm described above is used to separate a specific probe from the surface of the microsphere or to place the specific probe in the pro Sequence in an aqueous environment.
  • a spacer arm sequence of appropriate length between the probe sequence and the amino group By arranging a spacer arm sequence of appropriate length between the probe sequence and the amino group, steric hindrance can be reduced, the efficiency of the hybridization reaction, and the specificity of the hybridization reaction can be improved.
  • Common spacer sequences include poly dT, poly (dT), oligotetraethylene glycol, and (CH2) n spacer arms (n ⁇ 3), such as (CH2) 12, (CH2) 18 and the like.
  • poly (dA) interference is present, poly (TTG) can also be used as the spacer arm.
  • the spacer arm of the present invention is preferably 5-30 T, more preferably 10 D.
  • composition of amino-modified probes with spacer arms SEQ ID NO.
  • the primer for amplifying a target sequence having a 19-exon mutation site comprises a primer sequence of SEQ ID N0.13 to SEQ ID NO. 15, wherein at least one of the primers has a terminal biotin a marker; and/or a primer for SEQ ID N0.16 to SEQ ID N0.18 for amplifying a target sequence having a 21 exon mutation site, wherein at least one of the primers has a terminal biotin label.
  • Another object of the present invention is to provide a method for detecting mutation of an EGFR gene, which is rapid, accurate, and simple in operation, and can simultaneously detect multiple mutation sites in parallel.
  • the sample to be tested can be a tissue sample or a serum. Plasma or pleural effusion.
  • a method for detecting a mutation in a liquid phase chip EGFR gene using the above EGFR gene mutation detection comprising the following steps:
  • the second round of PCR amplification products are hybridized with the above-mentioned microspheres coated with the probe sequence; (5) After the hybridization reaction, streptavidin-phycoerythrin was added for reaction, and then the signal was detected by a Luminex apparatus.
  • the first round of PCR amplification is performed as: SEQ ID NO. 13, SEQ ID NO. 14 or SEQ ID NO. 16, SEQ ID NO. 17; performing a second round of PCR amplification
  • the pair of bows used SEQ ID NO. 13, SEQ ID NO. 15 and/or SEQ ID N 0.18, SEQ ID N 0.17.
  • the temperature of the hybridization is 55-60 °C.
  • the EGFR gene mutation detecting liquid phase chip provided by the invention can simultaneously detect a site with a relatively high frequency of EGFR gene mutation, and can detect 19 exons and 21 exons separately, or Simultaneous detection of both, uniform reaction conditions during detection, good detection results, high sensitivity, detection accuracy of more than 90%.
  • the detection method of the present invention has simple steps, thereby avoiding many uncertain factors existing in a complicated operation process, and thus the detection accuracy can be greatly improved.
  • the detection method provided by the present invention requires much less time than the commonly used sequencing technology, and is particularly in line with clinical needs.
  • the present invention adopts a method of enzymatic digestion and enrichment to carry out PCR amplification of a target sequence and is used for detection, thereby avoiding interference caused by a large number of wild-type sequences in the product.
  • the detection method provided by the invention not only can detect the EGFR gene mutation in the tumor tissue sample, but also can detect the EGFR gene mutation in the body fluid of the tumor patient, thereby having the advantages of convenient sampling, small patient pain, and dynamic monitoring. detailed description
  • Polystyrene microspheres were used as the reaction carrier, and the fluorescence detector was used as a detection platform to perform high-throughput multi-index parallel detection of nucleic acid molecules.
  • different proportions of red and infrared light dyeing agents are incorporated to form up to 100 different color coded microspheres.
  • Different microspheres covalently bind nucleic acid molecules for different analytes as probe molecules, reporter molecules are labeled with biotin, and stained with highly sensitive fluorescent dyes.
  • These microspheres and the analyte, reporter, and fluorescent label form a complete microsphere detection system for reading in the Luminex system.
  • the Luminex reading system inspires the red laser and the green laser to detect the microsphere system, respectively.
  • the red laser detects the intensity of the red-classified fluorescence on the surface of the microsphere and numbers it according to the different colors in the microsphere to determine the type of reaction;
  • the fluorescence intensity of the fluorescent label in the sample is detected, and the type and quantity of the microspheres detected by the laser are automatically statistically analyzed by a machine and a computer to determine the respective concentrations of the plurality of target test objects of the sample to be tested.
  • Washing solution 0.2ml/L Tween-20 (Sigma P-9416), lg/L SDS (Sigma L-4390)
  • TE pH 8.0
  • stock solution lOmmol/L Tris (Sigma 337501), lmmol/L EDTA (Sigma E-5134) 2xTm hybridization buffer Reagent source final concentration per 250ml lMTris-HCl, pH 8.0 SigmaT3038 0.2M 50ml
  • oligonucleotide probes were designed for wild-type and mutant sequences of the 19, 21 exons of the EGFR gene.
  • the 5' end of the probe is an amino group followed by a 10 T spacer arm.
  • the probe was synthesized by Shanghai Biotech Engineering Services Co., Ltd.
  • the probes were coupled together by covalent bonding with different color-coded microspheres (purchased from Luminex) (coating process).
  • the probe sequences are shown in the following table:
  • the microspheres were tested for the effect of detecting the microspheres containing the wild type probe in the first reaction system and the corresponding complementary sequence of the biotin label for the coating effect detection, ie E19w- P+E19w-b;
  • the second set of reaction system contains wild-type probe-coated microspheres, the mutant probe-coated microspheres and the reverse complement of the biotin-labeled wild-type probe, ie E19w-P +E19m-P+E19w-b ;
  • the reverse complement of the mutant probe-coated microspheres and the biotinylated wild-type probe in the third reaction system is E19W-P+E19m-b;
  • the reverse complementary sequence of the mutant probe-coated microspheres and the biotin-labeled mutant probe in the reaction system is E19m-P+E19m-b;
  • the fifth group reaction system contains the mutant probe coated The reverse complement of the
  • the experimental results show that the 19 exon wild type probe, 19 exon mutant probe, 21 exon wild type probe and 21 exon mutant probe designed by the invention can be well recognized.
  • the fluorescence values after hybridization are all above 2500.
  • the cross-reaction rate is within 10%, which basically does not affect the test results.
  • the primers were synthesized by Shanghai Shenggong Bioengineering Technology Co., Ltd. Among them, Exonl9 - Sl and Exonl9 - Asl are used The first round of PCR amplification of exon 19, biotin-labeled Exonl9-Sl and Exonl9-As2 were used for the second round of PCR amplification of exon 19; Exon21-S1 and Exon21-Asl were used for exon 21 For the first round of PCR amplification, Exon21-Asl and biotinylated Exon21-S2 were used for the second round of PCR amplification of exon 21.
  • the prepared liquid phase chip for detecting EGFR gene mutation includes:
  • Microspheres of the del E746-A750(1) mutant amino-modified probe of Exonl9 exon of SEQ ID NO. 6, coated with del E746-A750 of Exonl9 exon of SEQ ID N0.31 Microspheres of a mutant amino-modified probe, microspheres coated with a wild-type amino-modified probe against Exon21 exon of SEQ ID N0.9, and coated with SEQ ID N0.12 a microsphere of a mutant amino-modified probe of the ⁇ exon, each of the above microspheres having a different color code;
  • the reaction system is as follows:
  • the enzyme was incubated at 37 ° C for 2 hours and at 65 ° C for 20 minutes.
  • the reaction system is as follows:
  • the first round of PCR amplification and digestion is the same as above, except that the second round of PCR amplification adopts simultaneous amplification.
  • Biotin-Exon 19 SI 0.5 ⁇ 1 (20 ⁇ 1/ ⁇ )
  • the mixed microsphere working fluid will be vortex 30s, sonicated for 30s;
  • the EGFR gene mutation detection liquid phase chip and the detection method thereof provided by the invention can accurately detect the deletion mutation of the exon 19 of the EGFR gene and the point mutation of the exon 21, and the accuracy rate is as high as 95%.
  • targeted drugs such as fentanib and erlotinib provides an important means of testing for clinically accurate drug use, avoiding aging loss of unnecessary treatment and economic loss.
  • Extraction of DNA from lung cancer tissue samples Take 5-50 mg of tissue specimens after lung cancer biopsy or biopsy. After grinding, wash twice with PBS solution of pH 7.4; Wash the tissue specimens and resuspend in 1 ml of digestive juice (50 mmol/ L Tris, lmmo/L Na 2 EDTA, 0.5% Tween-20, 200 ug/ml proteinase K 200, pH 8.5), digested in a 55 ° C water bath for 1 hour, 99 ° C water bath for 15 min inactivated proteinase K; 12000 rpm The mixture was centrifuged for 10 minutes; the supernatant was taken, and extracted by a phenol-chloroform-isoamyl alcohol method, and a DNA sample for a PCR reaction was obtained by an ethanol precipitation method. DNA can also be extracted by microcentrifugation column method; 2. PCR amplification and enzymatic digestion of the sample to be tested:
  • E19w-Pl Two sets of oligonucleotide probes (E19w-Pl, E19m-Pl, E19m-P13, E21w-Pl, E21m-Pl and E19w-P2) were designed for the wild type and mutant sequences of the EGFR gene 19 and 21 exons.
  • E21W-P2, E21m-P2) The 5, end of the 0 probe is an amino group, followed by a 10 T spacer arm.
  • the probe was synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. The probes were coupled together with co-valent binding to different color-coded microspheres (available from Luminex Corporation), respectively, as described in Example 1.
  • Microspheres coated with a wild-type amino-modified probe for Exonl9 exon of SEQ ID NO. 2, a mutant amino-modified with SEQ IDN0.5 for Exonl9 exon delE746-A750 (1) Microspheres of the probe, microspheres coated with a mutant amino-modified probe of SEQ ID NO. 30 against Exonl9 exon del E746-A750 (2), coated with Exon21 outside SEQ ID N0.8 Microspheres of a wild-type amino-modified probe of the exon, and microspheres of a mutant amino-modified probe coated with Exon21 exon of SEQ ID NO. 11 , each of the above microspheres Have different color coding; and
  • Example 2 Five to five non-small cell lung cancer serum samples used in Example 2 were selected for detection. The preparation of the sample to be tested, the PCR amplification and enzymatic digestion of the sample to be tested, the experimental steps of hybridization and on-machine detection are the same as those described in Example 2. The experimental results are as follows:
  • the liquid phase chip for detecting EGFR gene mutation in the present embodiment is used for detecting serum samples of lung cancer using E19w-P2, E19m-P2, E19mP12, E21w-P2, E21m-P2 probe coated microspheres for non-small Detection of serum samples of cell lung cancer.
  • a total of 1-5 serum samples from five non-small cell lung cancers were also selected for testing.
  • the preparation of the sample to be tested, the PCR amplification and enzymatic digestion of the sample to be tested, the experimental steps of hybridization and on-machine detection are the same as those described in Example 2.
  • the experimental results are as follows: Table 7 Test results of serum samples
  • the experimental results show that the probes E19w-P2, E19m-P2, E19m-P12, E21w-P2, E21m-P2 can also be used for the detection of mutations in clinical samples of EGFR, and the results of analysis and probes (E19w-P, E19m-P, E19m-Pl l , E21w-P, E21m-P) The results were consistent.

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Abstract

La présente invention concerne des sondes, des produits à base de puce liquide et des procédés pour la détection de mutations du gène EGFR. La puce liquide pour la détection de mutations du gène EGFR comprend principalement : des microsphères enrobées avec des sondes; des amorces pour amplifier une séquence cible dans l’exon 19 et/ou l’exon 21. La puce liquide et les procédés peuvent détecter des mutations avec une fréquence relativement plus élevée dans le gène EGFR, et détecter des mutations dans l’exon 19 et 21 simultanément ou séparément. L’essai a une spécificité et une sensibilité élevées avec des conditions de réaction uniformes. La détection a une exactitude supérieure à 90 pour cent et requiert moins de temps. Le procédé peut être appliqué non seulement à la détection de mutations du gène EGFR dans des échantillons de tissu tumoral mais également à celles dans des échantillons humoraux de patients cancéreux. Par conséquent, elle permet une surveillance dynamique.
PCT/CN2009/000429 2008-04-23 2009-04-22 Sondes, produits à base de puce liquide et procédés pour la détection de mutations du gène egfr WO2009129693A1 (fr)

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CN 200810184121 CN101445829B (zh) 2008-04-23 2008-12-08 Egfr基因突变位点的检测探针、液相芯片及其检测方法

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CN102234683B (zh) * 2010-04-23 2013-07-17 广州益善生物技术有限公司 一种egfr基因突变检测液相芯片
CN102181537B (zh) * 2011-03-22 2013-06-19 宁波基内生物技术有限公司 用于检测人类egfr基因21号外显子突变的引物组合物、试剂盒及方法
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